High-resolution multiphoton tomography of human skin in vivo and in vitro

The novel compact femtosecond NIR (near infrared) laser imaging system DermaInspect was used to perform for the first time in vivo high resolution non-invasive 4D tomography of human skin based on multiphoton autofluorescence imaging and second harmonic generation (SHG). Using fast galvoscan mirrors, a time correlated single photon counting (TCSPC) module and femtosecond 80 MHz laser pulses in the spectral range of 750 nm-850 nm human skin was analyzed with subcellular spatial resolution (3D) and 250 ps temporal resolution (4D). The non-linear induced autofluorescence originates from naturally endogenous fluorophores and protein structures like NAD(P)H, flavins, phorphyrins, melanin, elastin and collagen. Collagenous structures were detected using SHG. Tissues of patients with dermatological disorders like nevi and melanoma have been investigated with a clear visualization of cells and intratissue structures. Further characterization of those components was performed by the fluorescence lifetime imaging (FLIM) and the determination of two photon excitation spectra. This method of non invasive high resolution optical biopsy provides a painless diagnostic tool for dermatological applications.

[1]  Axel Bergmann,et al.  Picosecond fluorescence lifetime microscopy by TCSPC imaging , 2001, SPIE BiOS.

[2]  Gerald W. Lucassen,et al.  Two-photon fluorescence microscopy of in-vivo human skin , 2000, European Conference on Biomedical Optics.

[3]  Peter Fischer,et al.  Optical tomography of human skin with subcellular spatial and picosecond time resolution using intense near infrared femtosecond laser pulses , 2002, SPIE BiOS.

[4]  Klaus Hoffmann,et al.  Fluorescence Studies of Melanin by Stepwise Two-Photon Femtosecond Laser Excitation , 2000, Journal of Fluorescence.

[5]  William A Mohler,et al.  Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues. , 2002, Biophysical journal.

[6]  B R Masters,et al.  Multiphoton Excitation Microscopy of In Vivo Human Skin: Functional and Morphological Optical Biopsy Based on Three‐Dimensional Imaging, Lifetime Measurements and Fluorescence Spectroscopy a , 1998, Annals of the New York Academy of Sciences.

[7]  R Masters Barry,et al.  Three-Dimensional Confocal Microscopy of Human Skin In Vivo : Autofluorescence of Normal Skin , 1996 .

[8]  R. Webb,et al.  In vivo confocal scanning laser microscopy of human skin II: advances in instrumentation and comparison with histology. , 1999, The Journal of investigative dermatology.

[9]  Andreas Volkmer,et al.  Femtosecond Two‐photon Excited Fluorescence of Melanin* , 1999, Photochemistry and photobiology.

[10]  Guy Cox,et al.  3-dimensional imaging of collagen using second harmonic generation. , 2003, Journal of structural biology.

[11]  R. Webb,et al.  Video-rate confocal scanning laser microscope for imaging human tissues in vivo. , 1999, Applied optics.

[12]  B. Tromberg,et al.  Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence , 2002, Proceedings of the National Academy of Sciences of the United States of America.